Modification of post-trogocytosis effects to Enhance CAR-t cell persistence

Abstract

Abstract Trogocytosis is currently defined as a rapid membrane exchange process that occurs during cell-cell contact. Although widely reported in innate and adaptive immunity, its role in chimeric antigen receptor T (CAR-T) cell dysfunction has not been fully elucidated in acute myeloid leukemia (AML) background. In this study, we found that anti-CD33 CAR-T cells (CAR33) actively captured membrane components and intact CD33 proteins from AML cells via trogocytosis. Using fluorescence-based membrane labeling and confocal microscopy, we confirmed that CAR33 T cells captured membrane components within minutes of contact in an antigen-dependent manner. This process was actin cytoskeleton-dependent and correlated with antigen density when tested across CD33-expressing AML cell lines. Notably, the trogocytosed CD33 molecules retained their intracellular domains, as shown by the transfer of CD33-miRFP fusion proteins, indicating intact protein acquisition. Although CD33 is traditionally considered a myeloid lineage marker, we demonstrated that trogocytosed CD33 on CAR-T cells was functionally active. Specifically, trogocytosed CD33 initiated an inhibitory signaling cascade by recruiting the phosphatase SHP-1. This led to decreased phosphorylation of CD3ζ and ZAP70, impaired T cell receptor signaling, and diminished CAR-T proliferation. These findings highlight an underappreciated mechanism by which antigen acquisition through trogocytosis contributes to CAR-T dysfunction in AML. To address the limitations imposed by trogocytosed CD33 without directly disrupting trogocytosis, we engineered a modified CAR (CAR33M) construct by fusing a truncated form of suppressor of cytokine signaling 3 (SOCS3) to selectively target trogocytosed CD33 for accelerated proteasome-mediated degradation. CAR33M T cells exhibited enhanced proliferation, reduced apoptosis, a favorable memory phenotype, diminished expression of exhaustion markers, and elevated secretion of effector cytokines. In AML xenograft models, CAR33M T cells showed superior tumor clearance, prolonged persistence, and improved survival compared to conventional CAR33 T cells. Upon tumor rechallenge, CAR33M T cells underwent robust in vivo expansion, indicative of durable immune memory. Transcriptomic and protein phosphorylation analyses revealed selective enhancement of STAT5 signaling in CAR33M T cells. Collectively, this study identifies trogocytosed CD33 as a previously unrecognized inhibitory mediator in CAR-T cell signaling, and presents a rational engineering strategy to overcome trogocytosis-induced suppression and enhance CAR-T efficacy against AML.